Synergistic stabilization of a double mutant in chymotrypsin inhibitor 2 from a library screen in E. coli.
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ABSTRACT:
SUBMITTER: Hamborg L
PROVIDER: S-EPMC8373930 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:We have constructed mutants of chymotrypsin inhibitor 2 with short glutamine repeats inserted into its inhibitory loop. These mutants oligomerize when expressed in Escherichia coli. The dimer of a mutant with four glutamines now has been crystallized, and its structure has been solved by molecular replacement by using the wild-type monomer as a search model. The structure of each half of the dimer is found to be the same as that of the wild-type monomer, except around the glutamine insertion. It was proposed that the components of the oligomers are held together by hydrogen bonds between the main-chain and side-chain amides of the glutamine repeats. Instead, they appear to form by swapping domains on folding in E. coli, and the glutamine repeats connecting the components of the dimers are disordered.
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Project description:Control of human visceral leishmaniasis in regions where it is endemic is hampered in part by limited accessibility to medical care and emerging drug resistance. There is no available protective vaccine. Leishmania spp. protozoa express multiple antigens recognized by the vertebrate immune system. Since there is not one immunodominant epitope recognized by most hosts, strategies must be developed to optimize selection of antigens for prevention and immunodiagnosis. For this reason, we generated a cDNA library from the intracellular amastigote form of Leishmania chagasi, the cause of South American visceral leishmaniasis. We employed a two-step expression screen of the library to systematically identify T-cell antigens and T-dependent B-cell antigens. The first step was aimed at identifying the largest possible number of clones producing an epitope-containing polypeptide by screening with a pool of sera from Brazilians with documented visceral leishmaniasis. After removal of clones encoding heat shock proteins, positive clones underwent a second-step screen for their ability to cause proliferation and gamma interferon responses in T cells from immune mice. Six unique clones were selected from the second screen for further analysis. The corresponding antigens were derived from glutamine synthetase, a transitional endoplasmic reticulum ATPase, elongation factor 1gamma, kinesin K39, repetitive protein A2, and a hypothetical conserved protein. Humans naturally infected with L. chagasi mounted both cellular and antibody responses to these proteins. Preparations containing multiple antigens may be optimal for immunodiagnosis and protective vaccines.
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Project description:Breast Cancer Type 1 Susceptibility Protein (BRCA1)-deficient cells have compromised DNA repair and are sensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. Despite initial responses, the development of resistance limits clinical efficacy. Mutations in the BRCA C-terminal (BRCT) domain of BRCA1 frequently create protein products unable to fold that are subject to protease-mediated degradation. Here, we show HSP90-mediated stabilization of a BRCT domain mutant BRCA1 protein under PARP inhibitor selection pressure. The stabilized mutant BRCA1 protein interacted with PALB2-BRCA2-RAD51, was essential for RAD51 focus formation, and conferred PARP inhibitor as well as cisplatin resistance. Treatment of resistant cells with the HSP90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin reduced mutant BRCA1 protein levels and restored their sensitivity to PARP inhibition. Resistant cells also acquired a TP53BP1 mutation that facilitated DNA end resection in the absence of a BRCA1 protein capable of binding CtIP. Finally, concomitant increased mutant BRCA1 and decreased 53BP1 protein expression occur in clinical samples of BRCA1-mutated recurrent ovarian carcinomas that have developed resistance to platinum. These results provide evidence for a two-event mechanism by which BRCA1-mutant tumors acquire anticancer therapy resistance.
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Project description:Protein kinase D (PKD) has emerged as a potential therapeutic target in multiple pathological conditions, including cancer and heart diseases. Potent and selective small molecule inhibitors of PKD are valuable for dissecting PKD-mediated cellular signaling pathways and for therapeutic application. In this study, we evaluated a targeted library of 235 small organic kinase inhibitors for PKD1 inhibitory activity at a single concentration. Twenty-eight PKD inhibitory chemotypes were identified and six exhibited excellent PKD1 selectivity. Five of the six lead structures share a common scaffold, with compound 139 being the most potent and selective for PKD vs PKC and CAMK. Compound 139 was an ATP-competitive PKD1 inhibitor with a low double-digit nanomolar potency and was also cell-active. Kinase profiling analysis identified this class of small molecules as pan-PKD inhibitors, confirmed their selectivity again PKC and CAMK, and demonstrated an overall favorable selectivity profile that could be further enhanced through structural modification. Furthermore, using a PKD homology model based on similar protein kinase structures, docking modes for compound 139 were explored and compared to literature examples of PKD inhibition. Modeling of these compounds at the ATP-binding site of PKD was used to rationalize its high potency and provide the foundation for future further optimization. Accordingly, using biochemical screening of a small number of privileged scaffolds and computational modeling, we have identified a new core structure for highly potent PKD inhibition with promising selectivity against closely related kinases. These lead structures represent an excellent starting point for the further optimization and the design of selective and therapeutically effective small molecule inhibitors of PKD.
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Project description:Photosynthetic organisms provide food and energy for nearly all life on Earth, yet half of their protein-coding genes remain uncharacterized1,2. Characterization of these genes could be greatly accelerated by new genetic resources for unicellular organisms. Here we generated a genome-wide, indexed library of mapped insertion mutants for the unicellular alga Chlamydomonas reinhardtii. The 62,389 mutants in the library, covering 83% of nuclear protein-coding genes, are available to the community. Each mutant contains unique DNA barcodes, allowing the collection to be screened as a pool. We performed a genome-wide survey of genes required for photosynthesis, which identified 303 candidate genes. Characterization of one of these genes, the conserved predicted phosphatase-encoding gene CPL3, showed that it is important for accumulation of multiple photosynthetic protein complexes. Notably, 21 of the 43 higher-confidence genes are novel, opening new opportunities for advances in understanding of this biogeochemically fundamental process. This library will accelerate the characterization of thousands of genes in algae, plants, and animals.
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Project description:Despite significant therapeutic progress in multiple myeloma, drug resistance is uniformly inevitable and new treatments are needed. Our aim was to identify novel, efficacious small-molecule combinations for use in drug resistant multiple myeloma.A panel of 116 small molecule inhibitors was used to screen resistant myeloma cell lines for potential therapeutic targets. Agents found to have enhanced activity in the bortezomib or melphalan resistant myeloma cell lines were investigated further in combination. Synergistic combinations of interest were evaluated in primary patient cells.The overall single-agent drug sensitivity profiles were dramatically different between melphalan and bortezomib resistant cells, however, the bromodomain inhibitor, CPI203, was observed to have enhanced activity in both the bortezomib and melphalan resistant lines compared to their wild-type counterparts. The combination of bortezomib and CPI203 was found to be synergistic in both the bortezomib and melphalan resistant cell lines as well as in a primary multiple myeloma sample from a patient refractory to recent proteasome inhibitor treatment. The CPI203-bortezomib combination led to enhanced apoptosis and anti-proliferative effects. Finally, in contrast to prior reports of synergy between bortezomib and other epigenetic modifying agents, which implicated MYC downregulation or NOXA induction, our analyses suggest that CPI203-bortezomib synergy is independent of these events.Our preclinical data supports a role for the clinical investigation of the bromodomain inhibitor CPI203 combined with bortezomib or alkylating agents in resistant multiple myeloma.
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Project description:BackgroundMelanoma is the most aggressive and deadly form of skin cancer with increasing case numbers worldwide. The development of inhibitors targeting mutated BRAF (found in around 60% of melanoma patients) has markedly improved overall survival of patients with late-stage tumors, even more so when combined with MEK inhibitors targeting the same signaling pathway. However, invariably patients become resistant to this targeted therapy resulting in rapid progression with treatment-refractory disease. The purpose of this study was the identification of new kinase inhibitors that do not lead to the development of resistance in combination with BRAF inhibitors (BRAFi), or that could be of clinical benefit as a 2nd line treatment for late-stage melanoma patients that have already developed resistance.MethodsWe have screened a 274-compound kinase inhibitor library in 3 BRAF mutant melanoma cell lines (each one sensitive or made resistant to 2 distinct BRAFi). The screening results were validated by dose-response studies and confirmed the killing efficacies of many kinase inhibitors. Two different tools were applied to investigate and quantify potential synergistic effects of drug combinations: the Chou-Talalay method and the Synergyfinder application. In order to exclude that resistance to the new treatments might occur at later time points, synergistic combinations were administered to fluorescently labelled parental and resistant cells over a period of > 10 weeks.ResultsEight inhibitors targeting Wee1, Checkpoint kinase 1/2, Aurora kinase, MEK, Polo-like kinase, PI3K and Focal adhesion kinase killed melanoma cells synergistically when combined with a BRAFi. Additionally, combination of a Wee1 and Chk inhibitor showed synergistic killing effects not only on sensitive cell lines, but also on intrinsically BRAFi- and treatment induced-resistant melanoma cells. First in vivo studies confirmed these observations. Interestingly, continuous treatment with several of these drugs, alone or in combination, did not lead to emergence of resistance.ConclusionsHere, we have identified new, previously unexplored (in the framework of BRAFi resistance) inhibitors that have an effect not only on sensitive but also on BRAFi-resistant cells. These promising combinations together with the new immunotherapies could be an important step towards improved 1st and 2nd line treatments for late-stage melanoma patients.
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Project description:CRISPR knockout screening on MOLM-13 cells with midostaurin or gilteritinib
2020-01-09 | GSE143314 | GEO
Project description:Mutant IDH1 (mIDH1) inhibitors have shown single-agent activity in relapsed/refractory AML, though most patients eventually relapse. We evaluated the efficacy and molecular mechanism of the combination treatment with azacitidine, which is currently the standard of care in older AML patients, and mIDH1 inhibitor BAY1436032. Both compounds were evaluated in vivo as single agents and in combination with sequential (azacitidine, followed by BAY1436032) or simultaneous application in two human IDH1 mutated AML xenograft models. Combination treatment significantly prolonged survival compared to single agent or control treatment (P<.005). The sequential combination treatment depleted leukemia stem cells (LSC) by 470-fold. Interestingly, the simultaneous combination treatment depleted LSCs by 33,150-fold compared to control mice. This strong synergy is mediated through inhibition of MAPK/ERK and RB/E2F signaling. Our data strongly argues for the concurrent application of mIDH1 inhibitors and azacitidine and predicts improved outcome of this regimen in IDH1 mutated AML patients.
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Project description:Mesotrypsin is an unusual human trypsin isoform with inhibitor resistance and the ability to degrade trypsin inhibitors. Degradation of the protective serine protease inhibitor Kazal type 1 (SPINK1) by mesotrypsin in the pancreas may contribute to the pathogenesis of pancreatitis. Here we tested the hypothesis that the regulatory digestive protease chymotrypsin C (CTRC) mitigates the harmful effects of mesotrypsin by cleaving the autolysis loop. As human trypsins are post-translationally sulfated in the autolysis loop, we also assessed the effect of this modification. We found that mesotrypsin cleaved in the autolysis loop by CTRC exhibited catalytic impairment on short peptides due to a 10-fold increase in Km , it digested β-casein poorly and bound soybean trypsin inhibitor with 10-fold decreased affinity. Importantly, CTRC-cleaved mesotrypsin degraded SPINK1 with markedly reduced efficiency. Sulfation increased mesotrypsin activity but accelerated CTRC-mediated cleavage of the autolysis loop and did not protect against the detrimental effect of CTRC cleavage. The observations indicate that CTRC-mediated cleavage of the autolysis loop in mesotrypsin decreases protease activity and thereby protects the pancreas against unwanted SPINK1 degradation. The findings expand the role of CTRC as a key defense mechanism against pancreatitis through regulation of intrapancreatic trypsin activity.
| S-EPMC7076226 | biostudies-literature